Nucleic acid amplification technologies are widely used in clinical microbiology, blood screening, food safety, genetic disease diagnosis and prognosis, environmental microbiology, drug target discovery and validation, forensics, and other biomedical research. Robustness of nucleic acid amplification, specificity, sensitivity, reliability in terms of accuracy and precision, and affordability are of particular importance.
Nucleic acid sequence specific amplification allows sensitive detection of the presence of a specific sequence. Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are two thermocycling amplification technologies.
In contrast PCR and LCR, isothermal amplification refers to a category of amplification in which amplification is carried out at a substantially constant temperature. Transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), strand-displacement amplification (SDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA™), and exponential single primer isothermal amplification (X-SPIA™), self-sustained sequence replication (3SR) and loop mediated isothermal amplification (LAMP) are examples of isothermal amplification. Nucleic acid sequence can also be detected through signal amplification process, such as cycling probe and invader assay. Detectable signal is generated by nuclease cleavage of hybridized probe.
Because all enzymes, regardless its thermostability, are active in a range of temperature, such property could adversely affect nucleic acid amplification in terms of specificity, sensitivity and signal/noise ratio etc. This has been clearly demonstrated in PCR process. A thermostable DNA polymerase is essential for a PCR. Although optimal temperature of catalytic activity of a thermostable DNA polymerase is around 60˜75° C., it is also active at low temperature. It retains significant activity even at room temperature. Its activity at low temperature is a cause of primer dimer formation, non-specific amplification and reduced detection sensitivity.
Performance of DNA PCR is improved by employing hot-start technologies. “Hot start” refers to any method for assembling PCR reactions that keeps one or more of the reaction components physically or functionally separate from the rest of the components at low temperature and that allows the onset of the reactions at an elevated temperature. Hot-start PCR technologies are categorized into the following groups:                1. Physical barrier to divide all essential components into at least two compartments as disclosed in U.S. Pat. Nos. 5,411,876, 5,565,339; 5,413,924 and 5,643,764, all of which are incorporated herein by reference. The barrier is removed by heating at elevated temperature.        2. Reversible enzyme inhibitors to suppress enzyme activity at low temperature as disclosed in U.S. Pat. Nos. 5,338,671; 5,677,152; 5,773,258; 6,183,998; 5,693,502, 5,874,557, 5,763,173, 6,020,130, and 6,183,967, all of which are incorporated herein by reference. Binding of the inhibitor is either non-covalent or covalent. Hot-start by these methods is homogeneous and is the most widely used.        3. Phase separation of cofactor as disclosed in U.S. Pat. No. 6,403,341, incorporated herein by reference. Mg2+ is precipitated at low temperature and becomes soluble as temperature rises.        
One-step RT PCR is a process of amplifying RNA target by combining reverse transcribing RNA molecule and amplifying complementary DNA molecule in one vial. Target RNA molecules include HIV, HCV, West Nile Virus (WNV), human influenza virus, avian flu virus, Dengue virus, Ebola virus etc. In the United States, it is mandatory to test presence of HIV, HBV, HCV and WNV in donor blood. Performance of one-step RT PCR is critical to these clinical tests and blood screening. Unfortunately none of the existing hot-start technologies can be well applied to this process because:                1. Most reverse transcriptase, the key enzyme for reverse transcription, can't be a target for hot-start process because they are not thermostable and will lose activity after incubation at high temperature.        2. RNA molecule, the subject of the testing, is not stable and undergoes significant degradation at high temperature. Presence of divalent metal ion, such as Mg2+ makes the degradation much severer. None of the existing technologies could be applied without damaging target RNA molecules.        3. Long time incubation of reverse transcription process, usually 30 minutes or longer, tremendously increases chance of having side reactions that could reduce detection sensitivity dramatically. This shows that enzyme inhibitor based hot-start technology would not improve performance of one-step RT PCR.        
Because of practical importance of nucleic acid amplification, there is strong demand for a novel technology which can improve performance of the nucleic acid amplification reaction, especially one-step RT PCR In this application a novel controlled start of nucleic acid amplification reaction is described. All patents, patent applications, and publications mentioned herein, both supra and infra, are incorporated herein by reference.